Abstract: RNA interference (RNAi) provides defense against exogenous nucleic acids, such as viruses and transposons, in diverse organisms. The production of short interfering RNAs (siRNAs) antisense to the viral or transposon sequences is a hallmark of the RNAi response. The discovery of the endogenous antisense siRNAs (endo-siRNAs) matching thousands of protein coding sequences in C. elegans and identification of similar molecules in Drosophila and mammals poses a question about their function. Our recent microarray analysis of genes misregulated in the RNAi pathway mutants in C. elegans revealed preferential targeting by the RNAi components and endogenous short RNAs of genes whose inactivation is beneficial for stress resistance and lifespan extension, such as genes encoding translation factors. We propose that pools of endogenous short RNAs in C. elegans are subject to natural selection. Therefore, the composition of siRNAs in populations is adjusted in response to the environmental changes to achieve maximum fitness. The goal of this project is to test the above model. We will select populations of C. elegans resistant to specific environmental conditions and test these populations for the accumulation of endosiRNAs antisense to very specific genes whose inactivation allows survival under the tested condition. We already established a correlation between thermotolerance and accumulation of endo-siRNAs specific to translation initiation factors. In addition, natural selection for survival on the nematocidal drugs ivermectin and levamisole will be used to generate C. elegans strains resistant to drugs due to epigenetic endo-siRNA-based inactivation of specific genes. Proving the existence of a siRNA-based epigenetic natural selection would represent a fundamental breakthrough in basic science. Epigenetic RNAi-based mechanisms are not likely to be limited to lower organisms and may be involved in the immune escape and drug-resistance of malignant tumors and in other cases when cells evolve to escape the action of therapeutic agents. Public Health Relevance: This project is focused on revealing new mechanisms used by organisms for the adaptation to environment. If we confirm that composition of short RNA molecules in the nematode C. elegans is selected to maximize survival under specific conditions, this will open the doors for "natural selection" experiments aimed at generation of cells with specific characteristics. For example, neuronal cells with enhanced ability to resist oxidative stress could be generated.